PROJECT SUMMARY Nuclear pore complexes (NPCs) mediate the bi-directional transport of proteins, RNAs and ribonucleoprotein complexes across the double-membrane nuclear envelope of eukaryotic cells. Consequently, NPCs are essential for the ability of many biosynthetic, signaling and gene regulatory processes to maintain cellular health and viability. Protein mislocalization due to recognition defects or altered NPC structure and function is linked to diseases as diverse as primary biliary cirrhosis, amyotrophic lateral sclerosis (ALS), leukemias and cancers, and Alzheimer's and Huntington's diseases. While the protein components of the NPC and many soluble nuclear transport factors have been identified and extensively studied, the mechanism(s) by which bi-directional transport occurs without clogging the pore remains unknown. The NPC is an octagonally symmetric approximately cylindrical structure with an hourglass-shaped central pore that has an internal minimal diameter of ~50 nm in humans. Occluding this pore and decorating its exits is a network of > 200 mobile intrinsically disordered polypeptides. Thousands of phenylalanine-glycine (FG) repeat motifs within this disordered polypeptide network (FG-network) are binding sites for the nuclear transport receptors (NTRs) that carry cargos through the NPC. At steady-state, at least ~100 NTRs are asymmetrically distributed throughout the FG-network. The heterogeneous and dynamic NTR/FG-network establishes a permeability barrier while simultaneously providing pathways for the translocation of import and export complexes of a wide range of sizes, affinities and surface properties. Multiple preferred paths through the ~50 nm diameter pore are predicted for typical cargo complexes of ~5-10 nm. The extent of overlap in such pathways and the possibility of dynamic regulation remains largely unexplored due to the absence of technological tools to dissect these pathways with the necessary spatial and temporal resolution. To address this deficiency, three-dimensional (3D) super-resolution single molecule fluorescence microscopy and single particle tracking techniques will be used to explore various transport pathways to determine the extent of their structural and functional intersections. The Specific Aims are: 1) to develop a fast super-resolution 3D microscopy approach to characterize the translocation pathways through functional NPCs; and 2) to develop 3D distribution maps for NTRs and cargos undergoing transport. Aim 1 seeks to bring together multiple existing technologies to enable high precision 3D super-resolution microscopy on NPCs. Aim 2 seeks to then apply this technology to establishing a map of various transport pathways through the FG-network. This work will establish whether discrete transport pathways within the FG-network exist, and thus, enhance transport efficiency by minimizing interactions between import and export cargos. While the primary goal of the proposed work is to develop a comprehensive understanding of competing transport reactions and the potential implications for inhibition and regulation, the super-resolution microscopy technologies and algorithms developed are expected to comprise a necessary toolkit for the field as well as to have broad applicability.